B-factor values of CaV3.3 M1425 substitutions.

The T-type voltage-gated calcium channel CaV3.3 is expressed in GABAergic neurons of the thalamic reticular nucleus (TRN), where its pacemaking activity controls sleep spindle rhythmogenesis during the non-rapid eye movement (NREM) phase of natural sleep. Previously, we established CACNA1I, the gene coding for CaV3.3, as a disease gene for neurodevelopmental disease with or without epilepsy. Here we report three newly identified activation-gate-modifying heterozygous missense variants of CACNA1I, found in four unrelated patients with neurodevelopmental disease with or without seizures. One of these variants, p.(Met1425Val), is an amino-acid substitution at the same position as previously published variant p.(Met1425Ile). Notably, the other two variants studied here are also a pair of two different substitutions of the same amino acid: p.(Ala398Val) and p.(Ala398Glu). By using site-directed mutagenesis, voltage-clamp electrophysiology, computational modelling of neuronal excitability, and structure modelling, we found that the two substitutions of M1425 both result in a gain of channel function including left-shifted voltage-dependence of activation and inactivation, slowed inactivation and deactivation kinetics, and increased neuronal excitability. Remarkably, the two substitutions of A398 show opposite effects on channel function. While substitution A398E leads to a gain of channel function, A398V results in decreased current density, accelerated gating kinetics, and a decreased neuronal excitability. The lack of seizures in the two independent p.(Ala398Val) patients correlates with the absence of increased neuronal excitability in this variant. This is the first report of a gate-modifying CaV3.3 channel variant with partial loss-of-function effects associated with developmental delay and intellectual disability without seizures. Our study corroborates the role of CaV3.3 dysfunction in the etiology of neurodevelopmental disorders. Moreover, our data suggest that substantial gain-of-function of CaV3.3 leads to the development of seizures, whereas both gain- and loss-of-function variants of CACNA1I can cause neurodevelopmental disease.T型电压门控钙通道CaV3.3（T-type voltage-gated calcium channel）表达于丘脑网状核（thalamic reticular nucleus, TRN）的γ-氨基丁酸能神经元（GABAergic neurons）中，其起搏活动可调控自然睡眠非快速眼动（non-rapid eye movement, NREM）期的睡眠纺锤波节律发生。此前本课题组已明确编码CaV3.3的基因CACNA1I是伴或不伴癫痫的神经发育疾病的致病基因。本研究报道3个新发现的CACNA1I激活门控修饰型杂合错义变异，均见于4例伴或不伴癫痫发作的神经发育疾病无关患者。其中变异p.(Met1425Val)与已发表的p.(Met1425Ile)位于同一氨基酸位点。值得注意的是，本研究中另外两个变异为同一氨基酸位点的两种不同替换：p.(Ala398Val)与p.(Ala398Glu)。本研究通过定点诱变、膜片钳电生理学、神经元兴奋性计算建模及结构建模，发现M1425的两种替换均引发通道功能获得，具体表现为激活与失活的电压依赖性左移、失活及去激活动力学减慢，以及神经元兴奋性升高。令人意外的是，A398的两种替换对通道功能产生了相反效应：A398E可导致通道功能获得，而A398V则表现为电流密度降低、门控动力学加速及神经元兴奋性下降。2例独立的p.(Ala398Val)患者无癫痫发作，这与该变异未出现神经元兴奋性升高的表型相契合。本研究首次报道了一类兼具部分功能丧失效应、与发育迟缓和无癫痫发作的智力障碍相关的CaV3.3激活门控修饰型变异。本研究证实了CaV3.3功能异常在神经发育障碍病因学中的关键作用。此外，本研究数据提示，CaV3.3的显著功能获得会诱发癫痫发作，而CACNA1I的功能获得与功能丧失变异均可引发神经发育疾病。